The methylotrophic yeasts including Pichia pastoris have been widely used for production of recombinant proteins of commercial or medical importance. Many pharmaceutical compositions consist glycoproteins produced in methylotrophic yeasts including Pichia pastoris. However, production and medical applications of some therapeutic glycoproteins can be hampered by the differences in the protein-linked carbohydrate biosynthesis between these yeasts and the target organisms such as a mammalian subject.
Protein N-glycosylation originates in the endoplasmic reticulum (ER), where the precursor N-linked oligosaccharide of 14 sugars (Glc3Man9GlcNAc2) is assembled on a dolichol (a lipid carrier intermediate), and it is transferred to the appropriate Asn of growing nascent polypeptides. This is an event common to all eukaryotic N-linked glycoproteins. These glycans are then subjected to extensive modification as the glycoproteins mature and move through the ER via the Golgi complex to their final destinations inside and outside the cell. Three terminal glucose residues are trimmed away by glucosidase I and II, and one terminal α-1,2-linked mannose residue is removed by one or more different mannosidase in the ER, such as ER-mannosidase, resulting in the oligosaccharide Man8GlcNAc2. This glycoprotein is then transported to the Golgi apparatus where the sugar moiety undergoes various modifications. There are significant differences in the modifications of the sugar branches in the Golgi apparatus between yeasts and higher eukaryotes.
In mammalian cells, the modification of the sugar branches in the Golgi apparatus proceeds via three different pathways depending on the protein moieties to which the sugars are added. They are, (1) where the glycoprotein does not change; (2) where the glycoprotein is modified by adding the N-acetylglucosamine-1-phosphate moiety (GlcNAc-1-P) in UDP-N-acetyl glucosamine (UDP-GlcNAc) to the 6-position of mannose in the sugar branch, followed by removing the GlcNAc moiety to form an acidic sugar branch in the glycoprotein; or (3) where the N-linked glycan is first converted into Man5GlcNAc2 by removing three mannose residues by Golgi mannosidase I; Man5GlcNAc2 is further modified by adding one GlcNAc by N-acetylglucosamine transferase I (GlcNAc-Transferase I or GnTI) and removing two more mannose residues by mannosidase II. During subsequent terminal glycosylation there is addition of new terminal sugars including GlcNAc, galactose (Gal), fucose, and N-acetylneuraminic acid (also called sialic acid (NeuNAc)) to produce various hybrid or complex glycans (R. Kornfeld and S. Kornfeld, Ann. Rev. Biochem. 54: 631-664, 1985; Chiba et al J. Biol. Chem. 273: 26298-26304, 1998; Helenius A and Aebi M, Science 291:2364-2369, 2001).
In yeasts, the modification of the sugar branches in the Golgi apparatus involves a series of additions of mannose residues by different mannosyltransferases (“outer chain” glycosylation). The structure of the outer chain glycosylation is specific to the organisms, typically with more than 50 mannose residues in S. cerevisiae, and most commonly with structures smaller than Man14GlcNAc2 in Pichia pastoris. This yeast-specific outer chain glycosylation of the high mannose type is also denoted hyperglycosylation.
Hyperglycosylation is often undesired since it leads to heterogeneity of a recombinant protein product in both carbohydrate composition and molecular weight, which may complicate the protein purification. The specific activity (units/weight) of the hyperglycosylated proteins may be lowered by the increased portion of carbohydrate. In addition, the outer chain glycosylation is strongly immunogenic which is undesirable in a therapeutic application. Moreover, the large outer chain sugar can mask the immunogenic determinants of a therapeutic protein. For example, the influenza neuramimidase (NA) expressed in P. pastoris is glycosylated with N-glycans containing up to 30-40 mannose residues. The hyperglycosylated NA has a reduced immunogenicity in mice, as the variable and immunodominant surface loops on top of the NA molecule are masked by the N-glycans (Martinet et al. Eur J. Biochem. 247: 332-338, 1997).
Therefore, it is desirable to genetically engineer methylotrophic yeast strains in which glycosylation of proteins can be manipulated and from which recombinant glycoproteins can be produced having a mammalian-like glycosylation pattern.